Eleonora Franzè

Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth

Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

Involvement of interleukin-21 in the regulation of colitis-associated colon cancer

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane.

Characterization of IL-17A-Producing Cells in Celiac Disease Mucosa

Celiac disease (CD) is a gluten-sensitive enteropathy associated with a marked infiltration of the mucosa with IFN-γ–secreting Th1 cells. Recent studies have shown that a novel subset of T cells characterized by expression of high levels of IL-17A, termed Th17 cells, may be responsible for pathogenic effects previously attributed to Th1 cells. In this study, we characterized the expression of IL-17A–producing cells in CD. By real-time PCR and ELISA, it was shown that expression of IL-17A RNA and protein is more pronounced in active CD biopsy specimens in comparison with inactive CD and normal mucosal biopsy specimens. Flow cytometry confirmed that IL-17A is overproduced in CD mucosa and that CD4+ and CD4+CD8+ cells were major sources. The majority of IL-17A–producing CD4+ and CD4+CD8+ cells coexpressed IFN-γ but not CD161. The addition of a peptic‑tryptic digest of gliadin to ex vivo organ cultures of duodenal biopsy specimens taken from inactive CD patients enhanced IL-17A production by both CD4+ and CD4+CD8+ cells. Because we previously showed that IL-21, a T cell-derived cytokine involved in the control of Th17 cell responses, is overproduced in CD, we next assessed whether IL-17A expression is regulated by IL-21. Blockade of IL-21 activity by a neutralizing IL-21 Ab reduced IL-17A expression in cultures of active CD and peptic–tryptic digest of gliadin-treated CD biopsy specimens. In conclusion, our data show that IL-17A is increased in CD and is produced by cells that also make IFN-γ.

Inhibition of colitis by IL‐25 associates with induction of alternatively activated macrophages

Background: Interleukin (IL)‐25, a Th2‐related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL‐25, however, remains unknown. Since Th2‐cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL‐25‐mediated anticolitic effect. Methods: AAM‐related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6‐trinitrobenzenesulphonic acid (TNBS)‐induced colitis treated with IL‐25 and/or neutralizing IL‐4, IL‐13, and transforming growth factor beta 1 (TGF‐&bgr;1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL‐25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL‐25 on the alternative activation of peritoneal F4/80+ cells. Results: IL‐25 enhanced the expression of AAM‐related markers in F4/80+ cells infiltrating the peritoneum and colon of naïve and colitic mice. Peritoneal F4/80+ cells isolated from IL‐25‐treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL‐25 did not directly induce AAM in vitro and in vivo in mice, IL‐25 administration enhanced the expression of IL‐4, IL‐13, and TGF‐&bgr;1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL‐25‐driven AAM induction. Blockade of IL‐4, IL‐13, and TGF‐&bgr;1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL‐25 on AAM gene expression. Conclusions: The IL‐25‐mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties. (Inflamm Bowel Dis 2012;)

A functional role for Smad7 in sustaining colon cancer cell growth and survival.

Initially identified as an inhibitor of transforming growth factor (TGF)-β mainly owing to its ability to bind TGF-β receptor type I and abrogate TGF-β-driven signaling, Smad7 can interact with additional intracellular proteins and regulate TGF-β-independent pathways, thus having a key role in the control of neoplastic processes in various organs. Genome-wide association studies have shown that common alleles of Smad7 influence the risk of colorectal cancer (CRC), even though the contribution of Smad7 in colon carcinogenesis is not fully understood. In this study, we assessed the expression and role of Smad7 in human and mouse models of sporadic CRC. We document a significant increase of Smad7 in human CRC relative to the surrounding nontumor tissues and show that silencing of Smad7 inhibits the growth of CRC cell lines both in vitro and in vivo after transplantation into immunodeficient mice. Knockdown of Smad7 results in enhanced phosphorylation of the cyclin-dependent kinase (CDK)2, accumulation of CRC cells in S phase and enhanced cell death. Smad7-deficient CRC cells have lower levels of CDC25A, a phosphatase that dephosphorylates CDK2, and hyperphosphorylated eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, knockdown of Smad7 associates with inactivation of eIF2α, lower CDC25A expression and diminished fraction of proliferating cells in human CRC explants, and reduces the number of intestinal tumors in Apcmin/+ mice. Altogether, these data support a role for Smad7 in sustaining colon tumorigenesis.

IL-15 positively regulates IL-21 production in celiac disease mucosa

Celiac disease (CD)-associated inflammation is characterized by high interleukin- 21 (IL-21), but the mechanisms that control IL-21 production are not fully understood. Here we analyzed IL-21 cell sources and examined how IL-21 production is regulated in CD. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs), isolated from CD patients and non-CD controls, were analyzed for cell markers, cytokines, and transcription factors by flow cytometry. IL-21 was highly produced by CD4+ and CD4+/CD8+ IELs and LPLs in active CD. IL-21-producing cells coexpressed interferon-γ (IFN-γ) and to a lesser extent T helper type 17 (Th17) cytokines. Treatment of control LPLs with IL-15, a cytokine overproduced in CD, activated Akt and STAT3 (signal transducer and activator of transcription 3), thus enhancing IL-21 synthesis. Active CD biopsies contained elevated levels of Akt, and blockade of IL-15 in those samples reduced IL-21. Similarly, neutralization of IL-15 in biopsies of inactive CD patients inhibited peptic–tryptic digest of gliadin-induced IL-21 expression. These findings indicate that in CD, IL-15 positively regulates IL-21 production.

Tissue inhibitor of metalloproteinase-3 regulates inflammation in human and mouse intestine.

BACKGROUND & AIMS Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice. METHODS We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohns disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-β1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3-knockout (TIMP-3-KO), and transgenic (TIMP-3-Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3-KO T cells. RESULTS Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-β1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-β1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3-KO mice developed severe colitis after administration of TNBS, whereas TIMP-3-Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3-KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells. CONCLUSIONS TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-β1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.

Defective expression of SIRT1 contributes to sustain inflammatory pathways in the gut

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.

Plasma Cells in the Mucosa of Patients with Inflammatory Bowel Disease Produce Granzyme B and Possess Cytotoxic Activities

In both Crohn’s disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19+ and IgA+ cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19+ and IgA+ cells expressed perforin with no difference between IBD and controls. GrB-producing CD19+ cells expressed CD27 and were CD38high and CD20 negative. CD19+ B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19+ B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19+ and IgA+ cells, suggesting a role for these cells in IBD-associated epithelial damage.

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism.

Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)−25 in FH. IL‐25 expression was evaluated in patients with FH and in livers of mice with FH induced by D‐galactosamine (D‐Gal) and lipopolysaccharide (LPS). Mice were treated with IL‐25 before D‐Gal/LPS‐induced FH and before or after concanavalin A (ConA)‐induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL‐25 and analyzed for GR1+CD11b+ cells. CFSE‐labeled T cells were cocultured with GR1+CD11b+ cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti‐GR1 antibody before IL‐25 and D‐Gal/LPS administration. IL‐25 was constitutively expressed in mouse and human liver and down‐regulated during FH. IL‐25 prevented D‐Gal/LPS‐induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1+CD11b+ cells isolated from mice given IL‐25 inhibited T‐cell proliferation. Consistently, in vivo depletion of GR1+ cells abrogated the protective effect of IL‐25 in experimental D‐Gal/LPS‐induced FH. IL‐25 was both preventive and therapeutic in ConA‐induced FH. Conclusions: IL‐25 expression is markedly reduced during human and experimental FH. IL‐25 promotes liver accumulation of GR1+CD11b+cells with immunoregulatory properties. (Hepatology 2013;58:1436–1450)